Restoring biochemical activity and bacterial diversity in a trichloroethylene-contaminated soil: the reclamation effect of vermicomposted olive wastes

Background, aim, and scope  In this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylene-contaminated soil, which has previously been stabilized through vermicomposting processes, has been explored. Materials and methods  Trichloroethylene-contaminated water was pumped into soil columns with a layer of vermicompost at 10-cm depth (biobarrier system). The impacts of the trichloroethylene on the microbial community were evaluated by determining: (1) the overall microbial activity (estimated as dehydrogenase activity) and enzyme activities related to the main nutrient cycles (β-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-β-glucosidase complexes was performed to study the enzymatically active humic matter related to the soil carbon cycle. (2) The soil bacterial diversity and the molecular mechanisms for the bacterial resistance to organic solvents were also determined. For this, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to detect changes in bacterial community structure and PCR-single-strand conformational polymorphism (SSCP) was developed and optimised for detection and discrimination of the resistance-nodulation-division (RND) genes amplified from the contaminated soils. Results  Vermicompost reduced, with respect to the unamended soil, about 30% of the trichloroethylene leaching during the first month of the experiment. Trichloroethylene had a marked negative effect on soil dehydrogenase, β-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-β-glucosidase complexes that increased the extracellular activity related to C-cycle in the contaminated soils. The isoelectric focusing technique showed a more biochemically active humic matter in the soil sampled under the vermicompost. The behaviour of the three main phyla of bacteria isolated from the DGGE bands was quite different. Bands corresponding to Actinobacteria disappeared, whereas those affiliated with Proteobacteria remained after the trichloroethylene contamination. The disappeared Actinobacteria became visible in the soil amended with the vermicompost. Bands corresponding to Bacteriodetes appeared only in columns of contaminated soils. In this study, six types of RND proteins were detected by PCR-SSCP in the natural soil, three in the trichloroethylene-contaminated soil and 7/5 in trichloroethylene-contaminated soil above/below the vermicompost in the biobarrier columns. Trichloroethylene tended to reduce or eliminate all the clones detected in the uncontaminated soil, whereas new efflux pumps appeared in the biobarrier columns. Discussion  Although enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity in the amended soils. The decrease was less severe in the biobarrier system, but in any case, no relation was found between the levels of trichloroethylene in soil and extracellular β-glucosidase activity, or between the latter and the quantity of humic carbon in soils. The isoelectric focusing technique was carried out in the humic fraction to determine whether the loss of activity occurred in overall extracellular β-glucosidase or in that linked to stable humic substances (humic–enzyme complexes). The contaminated soils showed the lower enzyme activities, whereas contaminated and amended soils presented greater quantity of focalised (and therefore stable) humic carbon and spectra heterogeneity: very different bands with higher enzyme activities. No clear relationship between trichloroethylene concentration in soil and diversity of the bacterial population was noted. Similar patterns could be found when the community structures of bacteria and microbial activity were considered. Since the use of the dehydrogenase assay has been recognised as a useful indicator of the overall measure of the intensity of microbial metabolism, these results could be attributed to PCR-DGGE methodology, since the method reveals the presence of dominant populations regardless of their metabolic state. Trichloroethylene maintained or even increased the number of clones with the DNA encoding for RND proteins, except for the contaminated soil located above the vermicompost. However, the main effect of trichloroethylene was to modify the structure of the community in contaminated soils, considering the type of efflux pumps encoded by the DNA extracted from soil bacteria. Conclusions  Trichloroethylene inhibited specific functions in soil and had a clear influence on the structure of the autochthonous bacterial community. The organic matter released by the vermicomposted olive waste tended to avoid the toxic effect of the contaminant. Trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity, even when vermicompost was present. In this case, the effect of the vermicompost was to provide and/or to stimulate the humic-β-glucosidase complexes located in the soil humic fraction >10⁴, increasing the resistance of the enzyme to the inhibition. The bacterial community from the soil presented significantly different mechanisms to resistance to solvents (RND proteins) under trichloroethylene conditions. The effect of the vermicompost was to induce these mechanisms in the autochthonous bacterial community and/or incorporated new bacterial species, able to grow in a trichloroethylene-contaminated ambient. Coupled biochemical and molecular methodologies are therefore helpful approaches in assessing the effect of an organic amendment on the biochemical and biological restoration of a trichloroethylene-contaminated soil. Recommendations and perspectives  Since the main biochemical and biological effects of the organic amendment on the contaminated soil seem to be the incorporation of biochemically active humic matter, as well as new bacterial species able to grow in a trichloroethylene-contaminated ambient, isoelectric focusing and PCR-SSCP methodologies should be considered as parts of an integrated approach to determine the success of a restoration scheme.     

卤代酚复合污染对鲤科金鱼的生化毒性效应研究

为探讨卤代酚复合污染对水生生物的联合毒性效应,探究较为可靠的检测方法,根据所选6种卤代酚化学结构的相似程度和48 h LC₅₀值,将其分为3组二元混合物进行急性暴露实验, 毒性单位比为1∶1,每组分2个浓度级别观察金鱼生物学行为的变化,同时检测金鱼肝脏中3种抗氧化酶(SOD,CAT和GSH-Px)活性以及酶活性影响率的变化. 结果表明:3组卤代酚对金鱼肝脏中3种抗氧化酶的酶活性影响率变化均有显著性差异(P<0.01);3种抗氧化酶中仅SOD在1/2LC₅₀剂量暴露组中均表现为明显升高的酶活性激活率或酶活性抑制率,提示SOD较适合作为卤代酚低浓度复合水污染情况监测的生物化学检测指标.      

胞外多聚物在好氧颗粒污泥形成中的作用机制

在SBR反应器活性污泥好氧颗粒化过程中,分析不同时期污泥EPS主要成分的变化、污泥表面特性的变化及二者相关关系.不同时期污泥样品胞外蛋白的SDS-PAGE表明,蛋白分子量条带主要分布在（31.0～97.4）×10³,与种泥相比,颗粒污泥在形成过程中增加了一些新蛋白条带,且条带颜色逐渐变深,定性表明蛋白种类及含量的增加.定量测定也表明,胞外蛋白分泌量随颗粒污泥的形成逐渐从49.4 mg·g^-1增至148.3 mg·g^-1,多糖则无明显变化,蛋白/多糖值也从2.3逐渐增至4.9.颗粒污泥表面疏水性比种泥约增加1倍,疏水性的变化与蛋白/多糖值正相关,相关系数为0.969.测得种泥与颗粒污泥表面Zeta电位平均值分别为-28.5 mV和-13.2　mV,颗粒污泥表面的电负性明显降低.由蛋白质自身特性,可推测其通过增加污泥表面相对疏水性和降低污泥表面电负性而促进好氧颗粒污泥形成的作用机制.     

石墨烯/血红素仿生涂层促进电活性微生物产电的效应和机理研究

阳极输出效率是决定生物电化学系统(BESs)能否实现工程化应用的基础和关键.但微生物/阳极界面的低效电子传递阻碍了 BESs整体输出效率的进一步提升.本研究通过模拟细胞色素c构建了一种石墨烯/血红素(GN/Hemin)复合仿生材料修饰的高性能BESs阳极.通过监测电活性菌ShewanellaloihicaPV-4的电能...     

黑藻对十二烷基苯磺酸钠和多聚磷酸钠胁迫的响应

研究了在十二烷基苯磺酸钠(SDBS)和多聚磷酸钠(STPP)及其复合胁迫下,黑藻过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性变化及其耐受浓度.结果表明,POD活性随着处理浓度的增加而逐渐增大;CAT在低浓度胁迫时活性逐渐增加,在较高浓度胁迫时活性降低;实验初期(6h时),SOD活性逐渐升高,随着SDBS处理浓度的提高及时间延长,SOD活性下降,并且在8mg/L浓度处理72h,SOD失活.SDBS对黑藻SOD的抑制作用大于STPP.黑藻对SDBS的耐受浓度(8mg/L)明显小于STPP的耐受浓度(256mg/L).黑藻对SDBS和STPP复合胁迫的耐受浓度值为(32+64)mg/L.     

铜对夏腊梅种子萌发及抗氧化酶活性的影响

采用室内水培试验,研究不同浓度的外源铜处理对夏腊梅(Sinocalycanthus chinensis)种子萌发、根伸长抑制率、呼吸强度、丙二醛及抗氧化酶系统的影响.结果表明,(1)铜浓度与夏腊梅根伸长抑制率呈显著正相关(P＜0.01),但与萌发率相关性不显著.(2)铜处理对夏腊梅种子的过氧化物酶(POD EC1.11.1.7)具有激活效应,且随着铜浓度的增加激活效应逐渐增强.丙二醛(MDA)含量随铜浓度的增大先略降而后逐渐升高.对超氧化物歧化酶(SOD EC 1.15.1.1)和过氧化氢酶(CAT EC 1.11.1.6)存在胁迫初期低浓度下的激活效应,随着铜浓度的增加及胁迫时间的延长,表现为抑制效应,且随着铜浓度的增加抑制效应逐渐增强.     

苯酚-硫酸法测定好氧颗粒污泥胞外聚合物中多糖浓度的研究

以葡萄糖为对照，优选苯酚-硫酸法测定胞外聚合物中多糖含量的显色条件，对此法精密度进行研究。结果表明，此测定方法显色稳定，重复性好，但回收率欠佳，测得的多糖含量要大于实际值。     

Evaluation of antioxidant enzymes activities and identification of intermediate products during phytoremediation of an anionic dye (C. I. Acid Blue 92) by pennywort (Hydrocotyle vulgaris)

The potential of pennywort(Hydrocotyle vulgaris)for phytoremediation of C.I.Acid Blue 92(AB92)was evaluated.The efects of various experimental parameters including pH,temperature,dye concentration and plant weight on dye removal efciency were investigated.The results showed that the optimal condition for dye removal were pH 3.5 and temperature 25°C.Moreover,the absolute dye removal enhanced with increase in the initial dye concentration and plant weight.Pennywort showed the same removal efciency in repeated experiments(four runs)as that obtained from the first run(a 6-day period).Therefore,the ability of the plant in consecutive removal of AB92 confirmed the biodegradation process.Accordingly,a number of produced intermediate compounds were identified.The efect of treatment on photosynthesis and antioxidant defense system including superoxide dismutase,peroxidase and catalase in plant roots and leaves were evaluated.The results revealed a reduction in photosynthetic pigments content under dye treatments.Antioxidant enzyme responses showed marked variations with respect to the plant organ and dye concentration in the liquid medium.Overall,the increase in antioxidant enzyme activity under AB92 stress in the roots was much higher than that in the leaves.Nevertheless,no significant increase in malondialdehyde content was detected in roots or leaves,implying that the high efciency of antioxidant system in the elimination of reactive oxygen species.Based on these results,pennywort was founded to be a capable species for phytoremediation of AB92-contaminated water,may be efective for phytoremediation dye-contaminated polluted aquatic ecosystems.     

活性污泥处理偶氮染料废水过程中胞外聚合物特性研究

采用序批式反应器(SBR)考察了偶氮染料浓度对活性污泥的污染物去除性能及胞外聚合物(Extracellular polymeric substances,EPS)的影响.结果表明,COD、染料及营养物的去除率均随着进水染料浓度的增加而下降,但进水染料浓度对EPS的影响却呈现不同的趋势.当进水染料浓度为5 ～40 mg·L-1时,EPS含量随着染料浓度的增加而增加;当进水染料浓度超过40 mg·L-1时,EPS含量却随着染料浓度的增加而减少.染料的加入导致活性污泥EPS中蛋白质的含量增加,且其变化趋势与EPS变化一致;EPS中腐殖酸的浓度低于蛋白质浓度;而EPS中多糖的浓度最低,为8 mg·g-1.三维荧光光谱结果显示,不同染料浓度下EPS荧光吸收峰数量及位置相同,分别为类蛋白峰(λEx/λEm=240 nm/375 ～394 nm)和类富里酸类峰(λEx/λEm =270 nm/410～416 nm),但两个吸收峰的荧光强度不同.     

Flocculating characteristic of activated sludge flocs: Interaction between Al3+ and extracellular polymeric substances

Aluminum flocculant can enhance the flocculating performance of activated sludge.However,the binding mechanism of aluminum ion(Al 3+) and extracellular polymeric substances(EPS) in activated sludge is unclear due to the complexity of EPS.In this work,threedimensional excitation emission matrix fluorescence spectroscopy(3DEEM),fluorescence quenching titration and Fourier transform infrared spectroscopy(FT-IR) were used to explore the binding behavior and mechanism between Al 3+ and EPS.The results showed that two fluorescence peaks of tyrosineand tryptophan-like substances were identified in the loosely bound-extracellular polymeric substances(LB-EPS),and three peaks of tyrosine-,tryptophanand humic-like substances were identified in the tightly boundextracellular polymeric substances(TB-EPS).It was found that these fluorescence peaks could be quenched with Al 3+ at the dosage of 3.0 mg/L,which demonstrated that strong interactions took place between the EPS and Al 3+.The conditional stability constants for Al 3+ and EPS were determined by the Stern-Volmer equation.As to the binding mechanism,the-OH,N-H,C=O,C-N groups and the sulfurand phosphorus-containing groups showed complexation action,although the groups in the LB-EPS and TB-EPS showed different behavior.The TB-EPS have stronger binding ability to Al 3+ than the LB-EPS,and TB-EPS play an important role in the interaction with Al 3+.